• Biomolecules & Therapeutics
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• 제4권1호
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• Bulletin of the Korean Chemical Society
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• 제28권6호
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• pp.1005-1009
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• 2007

Leukotriene B4 (LTB4) is a potent chemoattractant for leukocytes and considered to be an inflammatory mediator. Human BLT2 (hBLT2) is a low-affinity G-protein coupled receptor for LTB4 and mediates pertussis toxin-sensitive chemotactic cell movement. Here, we dissected the interaction between hBLT2 and G-protein alpha subunits using GST fusion proteins containing intracellular regions of hBLT2 and various Gα protein including Gα i1, Gα i2, Gα i3, Gα s1, Gα o1, and Gα z. Among the tested Gα subunits, Gα i3 showed the highest binding to the third intracellular loop region of hBLT2 with a dissociation constant (KD) of 5.0 × 10?6 M. These results suggest that Gα i3 has the highest affinity to hBLT2, and the third intracellular loop region of hBLT2 is the major component for the interaction with Gα i3.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.59-59
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• 2003

Serotonin (5-HT; 5-hydroxytryptamine) exerts multiple effects on central nervous system as well as behaviors such as mood and appetite. The signaling of serotonin is mediated by 7 families of serotonin receptors, designated 5-HT$_1$ to 5-HT$_{7}$. Six families of this receptor are G-protein coupled 7TM receptors, and the third intracellular loop of these receptors is proposed to interact with specific types of G-proteins. To investigate the specific interaction between the third intracellular loop of 5-HT$_{6}$ with G$\square$s, we have constructed a chimera protein that represent the third intracellular loop of 5-HT$_{6}$ within a leucine zipper motifs, In addition an alpha subunit of human G-protein that interact with 5-HT$_{6}$ was cloned into a bacterial expression vector. The two proteins were expressed in E. coli and purified in homogeneity. The interaction of the prepared proteins was examined by ELISA assay. The affinity between the two proteins and effect of insertion mutations were discussed.ussed.d.

• 한국정밀공학회:학술대회논문집
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• 한국정밀공학회 2004년도 추계학술대회 논문집
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• pp.1236-1239
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• 2004

The object of this study is to develop a model of the cardiovascular system capable of simulating the short-term transient and steady-state hemodynamic responses such as hypotention and disequilibrium syndrome during hemodialysis or hemofiltration. The model consists of a closed loop 12 lumped-parameter representation of the cardiovascular circulation connected to set-point models of the arterial and cardiopulmonary baroreflexes and 3 compartmental body fluid and solute kinetic model. The hemodialysis model includes the dynamics of sodium, urea, and potassium in the intracellular and extracellular pools, fluid balance equations for the intracellular, interstitial, and plasma volumes. We have presented the results of many different simulations performed by changing a few model parameters with respect to their basal values.

• 약학회지
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• 제44권1호
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• pp.52-59
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• 2000

An exceptionally conserved sequence that is shared among most G protein-coupled neurotransmitter receptors is an aspartate-arginine-tyrosine triplet that is located at the second cytoplasmic domain. Using the ml subtype of muscarinic acetylcholine receptors as an example, a point mutation of the arginine residue at position 123 into asparagine was induced. This mutation resulted in a complete blockade of the carbachol-induced increases of PI hydrolysis and intracellular $Ca^2$$^{+}$ level, in spite of the expression of the wild-type and mutant receptors at similar concentrations in Chinese hamster ovary cells. In marked contrast, the muscarinic agonist carbachol induced concentration-dependent enhancement of the activity of NO synthase at mutant ml receptors although the enhancement was significantly smaller than at wild-type ml receptors. These data suggest that this highly conserved arginine residue plays an important role in coupling of muscarinic receptors to the second messenger systems and the presence of alternate mechanisms of activation of neuronal NO synthase which might be operative in the absence of large changes in the concentration of cellular $Ca^{2+}$.2+/.

• Animal cells and systems
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• 제16권1호
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• pp.1-10
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• 2012

Most living organisms synchronize their physiological and behavioral activities with the daily changes in the environment using intrinsic time-keeping systems called circadian clocks. In mammals, the key molecular features of the internal clock are transcription- and translational-based negative feedback loops, in which clock-specific transcription factors activate the periodic expression of their own repressors, thereby generating the circadian rhythms. CLOCK and BMAL1, the basic helix-loop-helix (bHLH)/PAS transcription factors, constitute the positive limb of the molecular clock oscillator. Recent investigations have shown that various levels of posttranslational regulation work in concert with CLOCK/BMAL1 in mediating circadian and cellular stimuli to control and reset the circadian rhythmicity. Here we review how the CLOCK and BMAL1 activities are regulated by intracellular distribution, posttranslational modification, and the recruitment of various epigenetic regulators in response to circadian and cellular signaling pathways.

• 한국동물생명공학회지
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• 제35권2호
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• pp.155-162
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• 2020

Equine follicle stimulating hormone receptor (eFSHR) has a large extracellular domain and an intracellular domain containing approximately 10 phosphorylation sites within the G protein-coupled receptor. This study was conducted to analyze the function of phosphorylation sties at the eFSHR C-terminal region. We constructed a mutant of eFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 641 (eFSHR-t641). This removed 10 potential phosphorylation sites from the C-terminal region of the intracellular loop. The eFSHR-wild type (eFSHR-wt) and eFSHR-t641 cDNAs were subcloned into the pCMV-ARMS1-PK2 expression vector. These plasmids were transfected into PathHunter CHO-K1 Parental cells expressing β-arrestin 2 enzyme acceptor fusion protein and analyzed for agonist-induced cAMP response. The cAMP response in cells expressing eFSHR-t641 was lower than the response in cells expressing eFSHR-wt. EC₅₀ values of eFSHR-wt and eFSHR-t641 were 1079 ng/mL and 1834 ng/mL, respectively. eFSHR-t641 was approximately 0.58-fold compared with that of eFSHR-wt. The maximal response in eFSHR-wt and eFSHR-t641 was 24.7 nM and 16.7 nM, respectively. The Rmax value of phosphorylation sites in eFSHR-t641 was also decreased to approximately 68.4% of that in eFSHR-wt. The collective data implicate that the phosphorylation sites in the eFSHR C-terminal region have a pivotal role in signal transduction in PathHunter CHO-K1 cells, and indicate that β-arrestin is involved in coupling the activated receptors to the internalization system.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.93-93
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• 1997

Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.

• BMB Reports
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• 제46권4호
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• pp.219-224
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• 2013

The cell-fate specification of the anchor cell (AC) and a ventral uterine precursor cell (VU) in Caenorhabditis elegans is initiated by a stochastic interaction between LIN-12/Notch receptor and LAG-2/Delta ligand in two neighboring Z1.ppp and Z4.aaa cells. Both cells express lin-12 and lag-2 before specification, and a small difference in LIN-12 activity leads to the exclusive expressions of lin-12 in VU and lag-2 in the AC, through a feedback mechanism of unknown nature. Here we show that the expression pattern of lag-1/CSL, a transcriptional repressor itself that turns into an activator upon binding of the intracellular domain of Notch, overlaps with that of lin-12. Site-directed mutagenesis of LAG-1 binding sites in lag-1 maintains its expression in the AC, and eliminates it in the VU. Thus, AC/VU cell-fate specification appears to involve direct regulation of lag-1 expression by the LAG-1 protein, activating its transcription in VU cells, but repressing it in the AC.

• 한국식품영양과학회지
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• 제32권3호
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• pp.437-443
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• 2003

선행연구에서 라이코펜이 HT-29세포의 증식을 억제하는 것을 관찰하였기 때문에 본 연구는 그 기전을 연구하기 위하여 수행되 었다. 라이코펜이 HT-29 세포의 사멸을 유도하는지 조사하기 위해서 여러 농도의 라이코펜이 포함된 배지에서 세포를 4일 동안 배양하였다. 라이코펜 농도의 증가에 따라 사멸되는 세포에서 나타나는 특징의 하나인 DNA fragmentation이 증가하는 것을 관찰하였다. Western blot을 수행하여 얻은 결과에 의하면 라이코펜이 IGF-IR, IRS-1, PI3K, Akt와 같은 IGF-IR pathway에 속하는 단백질의 수준을 감소시켰다. IGF-IR의 인산화를 유도하기 위해서 라이코펜이 포함된 배지에서 세포를 배양하고 IGF-I을 첨가하여 0, 5, 10, 60분간 배양한 다음 IGF-IR antibody를 이용하여 immunoprecipitation을 수행하였다. 라이코펜은 IGF-I에 의한 IGF-IR, IRS-1의 인산화와 IGF-IR와 PI3K의 결합을 감소하고 인산화된 Akt를 감소시켰다. 이와 같은 IGF-IR signaling의 억제는 이 대장암세포에 존재하는 IGF-II의 autocrine loop을 억제하는 원인이 될 수 있어, 라이코펜의 암세포증식을 억제하는 기전 중의 하나가 될 수 있다. 라이코펜은 토마토와 그 가공품에 많이 존재하는 물질로 자연적인 식사를 통해 많이 섭취할 수 있는 물질이다. 라이코펜의 항암 기전을 밝혀냄으로써 미래 암예방과 치료를 위한 중요한 기능성 영양소를 생산할 수 있는 기초를 마련해줄 수 있을 것으로 기대된다.